As has frequently been discussed in the published literature, for example, Thrombos. Diath. Hemorrhage, Vol. 23, Pages 170-181 (1970), the amoebocyte blood cells of members of the genus Limulus, and particularly Limulus polyphemus, the horseshoe crab, form clots when placed in contact with pyrogen such as bacterial endotoxin. These amoebocyte cells provide an effective blood clotting mechanism to an injured horseshoe crab, thereby preventing further proliferation and migration of bacteria into other parts of the body.
At the present time, in vivo pyrogen testing of parenteral solutions is performed in rabbits. Such a test program is very expensive and difficult to operate.
A considerable amount of research has been invested in the use of Limulus amoebocytes, after lysing them in water or the like to rupture the cells, as a substitute testing means for pyrogens in sterile products. One typical summary of such recent work with Limulus is found in the Bulletin of the Parenteral Drug Association, Vol. 27, No. 3, Pages 39-148, May-June, 1973.
Typically, the Limulus amoebocyte cells are lysed by placing them in distilled water, or by any other convenient means for rupturing the blood cells. Following this, the resulting solution is filtered and centrifuged, to remove solids such as cell wall fragments and the like, to yield a protein solution, commonly referred to as Limulus lysate. This protein solution (Limulus lysate) is conventionally used to detect bacterial endotoxin by bringing it into contact with the material to be tested and observing whether or not a clot of protein is formed which has certain minimum standards of stability.
One typical testing standard for stability of the clot is to invert the test tube in which the clot is formed by 180.degree.. If the clot remains intact, a positive endotoxin reaction is recorded. If the clot breaks up, or no intact clot is ever formed, a negative endotoxin reaction is recorded.
Pure Limulus lysate in sterile water tends to have a lower degree of sensitivity to the presence of endotoxin (no better than 1.56 nanograms of endotoxin per ml.) than the sensitivity of the U.S.P. rabbit test for large volume parenteral solutions (down to 0.097 nanogram of endotoxin per ml.) and thus is usually not suitable for pyrogen testing of such solutions. While certain additives have been previously provided to the Limulus lysate solution to improve the stability of the lysate and the like (E. Thye Yin, et al., Biochem. Biophys. Acta, 261 (1972), Pages 284-289), there has been no report of the sensitivity of any prior art Limulus lysate becoming equal to that of the U.S.P. rabbit test for large volume parenteral solutions. Calcium has been added as a "potentiator", without achieving the desired sensitivity (Marchalonis, et al., Journal of Molecular Biology 32(2), Pages 453-465 (1968) ).
In accordance with this invention, various additive materials are disclosed which increase the sensitivity of Limulus lysate, frequently to a level which equals and even can exceed the endotoxin sensitivity which is available by the U.S.P. rabbit test for large volume parenteral solutions. The capacity for Limulus lysate solution to precipitate in the presence of extremely low concentrations of endotoxin is achieved by providing to the solution a catalytic concentration of one or more of the following ingredients: imidazole, manganese ions, Cleland's reagent and other equivalent organic disulfhydryl compounds, strontium ions, barium ions, cysteine and other equivalent organic monosulfhydryl compounds, or lithium ions.
It is generally preferred for the materials specified above to be present in the Limulus lysate solution in the following concentrations, expressed in terms of moles per liter of lysate solution.
______________________________________ Imidazole (commercially 0.004 to 0.4 mole per liter, available from the and preferably 0.01 to 0.3 Eastman Kodak Co.) mole per liter. Manganese preferably in the Mn.sup.++ form- 0.005 to 0.1 mole per liter and preferably 0.01 to 0.04 mole per liter. Cleland's reagent 0.00005 to 0.0005 mole per (commercially available liter and preferably 0.0001 from Sigma Chemical Co., to 0.0003 mole per liter. St. Louis, Missouri) Strontium Ions (Sr.sup.++) generally 0.005 to 0.4 mole per liter and preferably 0.01 to 0.3 mole per liter. Barium Ions (Ba.sup.++) generally 0.005 to 0.3 mole per liter, and preferably in the presence of well- known "Tris" buffer to provide a pH of 7 to 10. Cysteine generally 0.005 to 0.3 mole per liter. Lithium Ions (Li.sup.+) generally 0.005 to 0.3 mole per liter and preferably 0.03 to 0.15 mole per liter. ______________________________________
Magnesium ions by themselves provide excellent sensitization of the Limulus protein if in the presence of no more than 0.1 mole per liter of sodium ions, which tend to act as a suppressant of the catalytic effect of magnesium and generally tend to suppress the sensitivity of the lysate protein to endotoxin, even in the presence of other catalysts. Preferably from 0.05 to 0.3 mole of Mg.sup.++ ions are present per liter of solution. It is also preferred to avoid the use of hydroxyalkylmines in conjunction with magnesium ions. In the Thye Yin article cited above, "Tris" buffer, which is a hydroxylamine, has been used in combination with magnesium ions in the presence of about 0.15 molar concentration of sodium ions. Both of these materials can suppress the excellent catalytic effect of magnesium ions.
Preferably, the imidazole catalyst of this invention is utilized in the Limulus lysate solution in combination with Li.sup.+ ions, Mg.sup.++ ions, [Thioglycollate].sup.- ions (which may be added in the form of an alkali metal thioglycollate such as sodium thioglycollate), or Sr.sup.++ ions. Combinations of these ions with imidazole, which appears to act as a buffering agent, provide a particularly high sensitivity on the part of the Limulus lysate to the presence of endotoxin. For example, in conjunction with the above preferred concentration range of imidazole, excellent and frequently improved results can be obtained by the addition of 0.005 to 0.3 mole of Li.sup.+ ions per liter of solution, and preferably from 0.02 to 0.15 mole of the Li.sup.+ ions per liter of solution. Similarly, 0.005 to 0.3 mole of Mg.sup.++ ions, and preferably from 0.01 to 0.1 mole of Mg.sup.++ ions can be added per liter of solution.
A particularly excellent combination is found when imidazole and Mg.sup.++ ions are added in their above preferred concentrations, and [thioglycollate].sup.- ions are likewise added in a concentration of from 0.005 to 0.008 mole per liter of solution. Particularly superior results are usually obtained when from 0.02 to 0.03 mole of Mg.sup.++ ions; 0.02 to 0.03 mole of imidazole; and 0.001 to 0.002 mole of [thioglycollate].sup.- ions are present per liter of solution.
Similarly, from 0.001 to 0.01 mole of [thioglycollate].sup.- ions may be combined with the above concentration ranges of imidazole for improved results. Also, from 0.005 to 0.3 mole of Sr.sup.++ ions and preferably from 0.02 to 0.15 mole of Sr.sup.++ ions, per liter of solution, may be added to an imidazole solution to improve the performance over imidazole alone.
The above positively-charged ions may be added to the solution as their respective chlorides, although there has been no experimental evidence to show that the particular anion selected is absolutely critical, except that one should avoid anions which would tend to cause unfavorable side reactions with the Limulus lysate protein, or which render the lithium, magnesium, or strontium cations insoluble, or the like.
The use of the term "catalyst" herein is not intended to imply that the agents of this invention are true catalysts in the precise chemical sense, that is, where catalysts are not considered to be reactants. In this invention, where the mechanism of the action of these "catalysts" on Limulus lysate is not precisely understood, it is possible that the materials or agents disclosed herein as being "catalysts" may also be reactants. Therefore, the terms "catalyst" or "sensitizing agent" are used herein to mean a substance capable of providing improved capacity (or sensitivity) for Limulus lysate solution to precipitate in the presence of extremely low concentrations of endotoxin.
Manganese ions, particularly the Mn.sup.++ ions, have also been found to be active to potentiate the sensitivity of Limulus lysate. The manganese ions are typically added in the form of manganese chloride, although, as stated above, no particular criticality of the anion has been noted except for the obvious need to avoid insolubilizing anions and those which cause unfavorable side reactions.
Cleland's reagent is a material which is readily commercially available, and which increases the sensitivity of Limulus lysate, particularly when used in the concentrations specified above. It is believed that other organic disulfhydryl compounds will also perform equivalently.
Stontium ions (Sr.sup.++) give particularly excellent results, particularly when used in the concentrations prescribed above. The chloride of strontium is typically used, but other anions are available as well, subject to the restrictions described above.
Barium ions (Ba.sup.++) are particularly useful in combination with the well-known and commercially available "Tris" buffer (tris [hydroxymethyl]aminomethane) and equivalent buffering agents, especially when the buffer is present in a concentration to provide a pH of about 7 to 10.
Cysteine, and equivalent sulfhydryl materials (i.e., other organic monosulfhydryl compounds such as glutathione, an alkali metal thioglycollate, or thiouracil) exhibit a capacity to sensitize Limulus lysate, particularly in the concentrations specified above. Satisfactory results have also been obtained when commercially available cysteine hydrochloride is neutralized to about pH 6 to 10 with an alkali material such as sodium hydroxide, potassium carbonate, tetrabutylammonium hydroxide or the like.
In particular, excellent results are obtained by the use in the Limulus lysate solution of 0.02 to 0.2 percent by weight of a sulfhydryl-containing compound such as cysteine or an alkali metal thioglycollate salt, plus from 0.2 to 2 percent by weight of a water soluble magnesium salt such as magnesium chloride. Once again, the anion of the magnesium salt is not seen to be critical, with the exceptions described previously. Sodium thioglycollate is also highly suitable as the sulfhydryl-containing compound.
Lithium ions, such as can be provided by lithium chloride or a similar lithium salt having a soluble, noninterfering anion, are also effective for increasing the sensitivity of the Limulus lysate, particularly when used in the concentrations specified above, and when used in combination with imidazole.
The catalytic or sensitizing agents of this invention are conveniently utilized by first dissolving them in distilled water in the desired concentrations. Thereafter, the resulting solutions can be used to reconstitute lyophilized (freeze-dried) Limulus lysate, by dissolving the Limulus lysate in the solution. This technique is advantageous, because the Limulus lysate is preferably stored in lyophilized condition. The improved Limulus lysate solutions of this invention are preferably prepared immediately prior to use, particularly because their increased sensitivity to endotoxin makes them more likely to precipitate upon the accidental contamination that often takes place over prolonged storage.
As used herein, the term "solution" is primarily intended to refer to a water solution, and typically a distilled, pyrogen-free water solution which avoids premature precipitation of the Limulus lysate. However, it is contemplated that other, non-aqueous solvents such as methanol, ethanol, isopropanol, acetone, ethylene glycol, or other water miscible solvents can be used, particularly in a mixture with water, if desired.